In light of the benefit that the rat model system have provided to our basic understanding of the biology of human disease, and the importance of the rat to emerging fields of genomics and stem cell biology, the NIH initiated the Rat Genome Program and the Rat Genome Database. The NIH sponsored meeting on Rat Model Priorities recommended that a National Rat Genetic Resource Center be established to support research on germ-line modifications in the rat, including ear-marked funding for the study of rat sperm cryopreservation to alleviate the storage and transportation problems, and to improve in vitro fertilization techniques in the rat. Unfortunately, the current state of the art in rat sperm cryopreservation is abysmal. An electronic search of the literature reveals only two papers published in the last 40 years that seeks to optimize the cryopreservation of rat sperm (Nakatsukasa et al. 2001; 2003). This work establishes the feasibility of rat sperm cryopreservation but makes no attempt to optimize the technology, understand the damage caused to the sperm by cryopreservation, or to characterize the cryopreservation process in this species. In this proposal, we will attempt to achieve three very important goals as it relates to sperm cryopreservation in general and rat sperm cryopreservation in particular. First, we will attempt to optimize rat sperm cryopreservation by optimizing the freezing rate based careful characterization of the water transport properties of the sperm during freezing and optimization of the cryoprotective media. Second, we will determine the damage done to the sperm, at the molecular and structural level, caused by cryopreservation process, and design and test methods to minimize this damage based on our understanding of the mechanism. Finally, we will standardize approaches to reduce injury and optimize cryopreservation outcomes. These approaches are reflected in the following 3 specific aims: Specific Aim 1: Determine the handling methods and CPA media most effective for rat sperm cryopreservation. Specific Aim 2: Determine the biophysical water transport properties of rat sperm plasma membrane during freezing. Specific Aim 3: Design and test strategies for protecting rat sperm from damage incurred by cryopreservation.